Bacteriophages or plasmids are required for the synthesis of toxins in several pathogenic bacteria. The primary goal of our study is to determine genetic and biochemical mechanisms which regulate toxinogenesis in such systems. The production of diphtherial toxin by C. diphtheriae will be used as a model for phage-directed toxin synthesis. The regulation of synthesis of cholera toxin in V. cholerae will be analyzed and compared with the diphtherial system, and the possible role of plasmids in determining production of cholera toxin will be investigated. Our goals in the analysis of the diphtherial system are as follows: 1) We will identify genes which regulate the production of diphtherial toxin by selecting mutants of C. diphtheriae C7 or its phage Beta tox plus which increase or decrease the yield of diphtherial toxin. 2) We will determine the types of regulation involved in toxinogenesis by characterizing the mutant and wild-type strains for properties such as dominance, recessiveness, constitutivity, inducibility, and repressibility. 3) Methods for quantitative assay of toxin-specific messenger RNA will be developed. 4) We will use simultaneous measurements of toxin-specific mRNA and of toxin production to distinguish transcriptional from translational control in the synthesis of diphtherial toxin. We will analyze the regulation toxinogenesis in V. cholerae in the following sequence: 1) Nontoxinogenic mutants will be selected following treatment of V. cholerae with mutagens or with agents capable of eliminating plasmids. 2) Genes which regulate toxinogenicity in V. cholerae will be mapped by bacterial recombination experiments. 3) Tox plus and tox minus variants will be compared for the presence of phages or plasmids which might determine toxinogenicity. 4) The contribution of the tox plus character to virulence of V. cholerae will be examined in experimental animals infected with isogenic strains of V. cholerae differing only in toxinogenicity.